Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions.

Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase.

14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.

Kinetic analysis of 14-3-3-inhibited Arabidopsis thaliana nitrate reductase.

Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in k(cat) and a decrease in the apparent K(M)(nitrate) (from 280 to 141 µM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1.